The plasma membrane calcium pump (PMCA) is one of the essential mechanisms to extrude calcium across the plasma membrane, thereby keeping and restoring a low cytosolic calcium concentration. In mammals, four genes encode separate isoforms of this pump, and this variability is further enhanced by alternative splicing. In total, there are more than 20 variants of the PMCA. These PMCA isoforms are differentially expressed in various tissues where they participate in cell type-specific calcium regulation. As is the case in many other cation-transporting ATPases, the reaction cycle of the PMCA includes the synthesis and hydrolysis of a phosphorylated intermediate. However, little is known about differences in the reaction cycle among different PMCAs. Also, during cation transport across the membrane, it is possible to distinguish an intermediate step in which the transporting ion is not accessible to either side of the membrane. This phenomenon is known as ion occlusion, and so far has not been characterized in the PMCA. To characterize the differences in the reaction cycle among different PMCAs, including the analysis of calcium occlusion, it is necessary to combine expertise both in the molecular biology of PMCAs and in the kinetic analysis of their reaction cycle. The proposed collaboration between the laboratory of Dr. Emanuel E. Strehler at the Mayo Clinic in Rochester, MN, and the laboratory of Dr. Juan P. Rossi at the University of Buenos Aires in Argentina provides a unique opportunity to study this important topic. This research will be done as an extension of NIH grant # RO1GM28835.